Methods and materials for the production of monomers for nylon-4/polyester production

ABSTRACT

This document describes biochemical pathways for producing 4-hydroxybutyrate, 4-aminobutyrate, putrescine or 1,4-butanediol by forming one or two terminal functional groups, comprised of amine or hydroxyl group, in a C5 backbone substrate such as 2-oxoglutarate or L-glutamate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 62/095,556, filed on Dec. 22, 2014, which is incorporated by reference herein in its entirety.

TECHNICAL FIELD

This invention relates to methods and materials for biosynthesizing one or more C4 building blocks. This invention relates to methods and materials for biosynthesizing one or more C4 building blocks such as 4-hydroxybutyrate, 4-aminobutyrate, putrescine, and 1,4-butanediol using one or more polypeptides having decarboxylase, dehydrogenase, synthase, reductase, or ω-transaminase activity, and recombinant hosts that produce such C4 building blocks.

BACKGROUND

Four carbon compounds such as 1,4-butanediol (also known as butane-1,4-diol or tetramethylene glycol), putrescine, 4-hydroxybutyrate, and gamma 4-aminobutyrate (GABA) are used, for example, for producing plastics and polymers. 1,4-butanediol is used, for example, as a solvent and for producing plastics, Spandex fibers, and polymers such as polyurethane. 1,4-butanediol can be produced from malic anhydride by the Davy process, from acetylene using Reppe chemistry, or from propylene oxide in a multi-step process. Putrescine is used to produce Nylon-4,6 by reacting with adipic acid. Putrescine typically is produced by hydrogenating succinonitrile. However, the methods to produce such compounds typically are energy intensive and/or produce large amounts of by-products.

SUMMARY

There is a need for sustainable and efficient methods for producing 1,4-butanediol, putrescine, 4-hydroxybutyrate, and 4-aminobutyrate This document is based at least in part on the discovery that it is possible to construct biochemical pathways for producing a four carbon chain backbone precursor via decarboxylation of 2-oxoglutarate or L-glutamate, and forming one or two functional groups, i.e., amine or hydroxyl, in the four carbon chain backbone precursor, leading to the synthesis of one or more of 4-hydroxybutyrate, 4-aminobutyrate, putrescine (also known as tetramethylenediamine), and 1,4-butanediol (hereafter collectively referred to as “C4 building blocks” and each of the compounds being a “C4 building block”). Succinate semialdehyde (also known as 4-oxobutanoic acid) can be produced as an intermediate to other products. 4-hydroxybutyrate and 4-hydroxybutyric acid, 4-oxobutanoic acid and 4-oxobutanoate, and 4-aminobutyrate and 4-aminobutanoic acid are used interchangeably herein to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled in the art that the specific form will depend on pH.

In one aspect, this document features a method of producing 1,4-butanediol. The method includes enzymatically converting 4-hydroxybutyrate to 1,4 butanediol using a carboxylate reductase and an alcohol dehydrogenase. The 4-hydroxybutyrate can be enzymatically synthesized from L-glutamate or 2-oxoglutarate. For example, 4-hydroxybutyrate can be enzymatically synthesized from L-glutamate by enzymatically converting L-glutamate to 4-aminobutyrate, enzymatically converting 4-aminobutyrate to succinate semialdehyde, and enzymatically converting succinate semialdehyde to 4-hydroxybutyrate. For example, L-glutamate can be enzymatically converted to 4-hydroxybutyrate using (i) a glutamate decarboxylase; (ii) a ω-transaminase; and (iii) a dehydrogenase selected from the group consisting of a 4-hydroxybutyrate dehydrogenase and a 5-hydroxyvalerate dehydrogenase. For example, 4-hydroxybutyrate can be enzymatically synthesized from 2-oxoglutarate by, for example, enzymatically converting 2-oxoglutarate to succinate semialdehyde and enzymatically converting succinate semialdehyde to 4-hydroxybutyrate. For example, 2-oxoglutarate can be enzymatically converted to succinate semialdehyde using a 2-oxoglutarate decarboxylase and/or succinate semialdehyde can be enzymatically converted to 4-hydroxybutyrate using a 4-hydroxybutyrate dehydrogenase or a 5-hydroxyvalerate dehydrogenase. 2-oxoglutarate also can be enzymatically converted to L-glutamate, L-glutamate can be enzymatically converted to 4-aminobutyrate, 4-aminobutyrate can be enzymatically converted to succinate semialdehyde, and succinate semialdehyde can be enzymatically converted to 4-hydroxybutyrate.

This document also features a method of producing 4-hydroxybutyrate. The method includes enzymatically synthesizing 4-hydroxybutyrate from L-glutamate. L-glutamate can be enzymatically converted to 4-aminobutyrate, 4-aminobutyrate can be enzymatically converted to succinate semialdehyde, and succinate semialdehyde can be enzymatically converted to 4-hydroxybutyrate.

This document also features a method of producing putrescine, said method comprising a) enzymatically converting 4-hydroxybutyrate to putrescine using a carboxylate reductase, an alcohol dehydrogenase, and at least one ω-transaminase, or b) enzymatically converting 4-aminobutyrate to putrescine using a carboxylate reductase and a ω-transaminase.

This document also features a method of producing putrescine. The method includes enzymatically converting 1,4 butanediol to putrescine using at least one alcohol dehydrogenase and at least one ω-transaminase.

In any of the methods, L-glutamate can be enzymatically converted to 4-aminobutyrate using a glutamate decarboxylase having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:14 or SEQ ID NO: 19.

In any of the methods, the carboxylate reductase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

In any of the methods, the alcohol dehydrogenase can be classified under EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184.

In any of the methods, the ω-transaminase can be classified under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, 2.6.1.48, EC 2.6.1.76, EC 2.6.1.82, or EC 2.6.1.96. For example, the ω-transaminase can have at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

This document also features a recombinant host that includes at least one exogenous nucleic acid encoding (i) a glutamate decarboxylase, (ii) a dehydrogenase selected from the group consisting of a 4-hydroxybutyrate dehydrogenase and a 5-hydroxyvalerate dehydrogenase, and (iii) a first exogenous ω-transaminase, the host producing 4-hydroxybutyrate.

The host further can include an exogenous carboxylate reductase, a second optional and a third optional exogenous ω-transaminase, and an exogenous alcohol dehydrogenase, the host further producing putrescine.

The host further can include an exogenous carboxylate reductase and an exogenous alcohol dehydrogenase, the host further producing 1,4-butanediol.

This document also features a recombinant host that includes at least one exogenous nucleic acid encoding (i) a 2-oxoglutarate decarboxylase, (ii) a dehydrogenase, (iii) a carboxylate reductase, and (iv) an alcohol dehydrogenase, the host producing 1,4-butanediol. The dehydrogenase can be selected from the group consisting of a 5-hydroxyvalerate dehydrogenase and a 4-hydroxybutyrate dehydrogenase.

This document also features a recombinant host that includes at least one exogenous nucleic acid encoding a carboxylate reductase and at least one ω-transaminase, the host producing putrescine. In some cases, the host includes two exogenous ω-transaminases. In some cases, the host further includes at least one exogenous alcohol dehydrogenase (e.g., two exogenous alcohol dehydrogenases).

A host further can include an exogenous 2-oxoglutarate decarboxylase and an exogenous dehydrogenase.

A host further can include an exogenous glutamate synthase, an exogenous glutamate decarboxylase, a second exogenous ω-transaminase, and a dehydrogenase.

In any of the recombinant hosts, the at least one exogenous ω-transaminase can have at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, or SEQ ID NO: 11.

In any of the recombinant hosts, the carboxylate reductase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:6.

In any of the methods, all or part of the method can be performed in a recombinant host by fermentation. The host can be subjected to a cultivation strategy under aerobic, anaerobic or, micro-aerobic cultivation conditions. The host can be cultured under conditions of nutrient limitation. The host can be retained using a ceramic hollow fiber membrane to maintain a high cell density during fermentation. The principal carbon source fed to the fermentation can derive from a biological feedstock. For example, the biological feedstock can be, or can derive from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste. The principal carbon source fed to the fermentation can derive from a non-biological feedstock. For example, the non-biological feedstock can be, or can derive from, natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.

Any of the recombinant hosts or any of the recombinant hosts used in any of the methods can be a prokaryote. The prokaryote can be from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogemim or Clostridium khlyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; from the genus Delftia acidovorans, from the genus Bacillus such as Bacillus subtillis; from the genes Lactobacillus such as Lactobacillus delbrueckii; from the genus Lactococcus such as Lactococcus lactis or from the genus Rhodococcus such as Rhodococcus equi.

Any of the recombinant hosts or any of the recombinant hosts used in any of the methods can be a eukaryote. The eukaryote can be from the genus Aspergillus such as Aspergillus niger; from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; from the genus Yarrowia such as Yarrowia lipolytica, from the genus Issatchenkia such as Issathenkia orientalis, from the genus Debaryomyces such as Debaryomyces hansenii, from the genus Arxula such as Arxula adenoinivorans, or from the genus Kluyveromyces such as Kluyveromyces lactis.

The recombinant host or recombinant host used in any of the methods can include one or more of the following attenuated enzymes: a polyhydroxyalkanoate synthase; a triose phosphate isomerase; a glucose-6-phosphate isomerase; a transhydrogenase; an NADH-specific glutamate dehydrogenase; or a NADH/NADPH-utilizing glutamate dehydrogenase.

Any of the recombinant hosts or any of the recombinant hosts used in any of the methods can overexpress one or more genes encoding: a phosphoenolpyruvate carboxylase; a pyruvate carboxylase; a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a formate dehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose dehydrogenase; a glucose-6-phosphate dehydrogenase; a fructose 1,6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase; a L-glutamine synthetase; a lysine transporter; a dicarboxylate transporter; and/or a multidrug transporter.

In one aspect, this document features a biochemical network comprising a carboxylate reductase and an alcohol dehydrogenase. 4-hydroxybutyrate, and 1,4 butanediol, wherein the carboxylate reductase and the alcohol dehydrogenase enzymatically convert 4-hydroxybutyrate to 1,4-butanediol. The biochemical network further can include a glutamate decarboxylase; a ω-transaminase; and a dehydrogenase selected from the group consisting of a 4-hydroxybutyrate dehydrogenase and a 5-hydroxyvalerate dehydrogenase, wherein the glutamate decarboxylase, the ω-transaminase, and the dehydrogenase enzymatically convert L-glutamate to 4-hydroxybutyrate.

This document also features a means for producing 1,4 butanediol, wherein the means enzymatically converts 4-hydroxybutyrate to 1,4 butanediol. The means can include a carboxylate reductase and an alcohol dehydrogenase.

This document also features a means for producing putrescine, wherein the means enzymatically converts 4-hydroxybutyrate to putrescine. The means can include a carboxylate reductase, an alcohol dehydrogenase, and at least one ω-transaminase.

In another aspect, this document features a step for obtaining 1,4-butanediol using a carboxylate reductase and an alcohol dehydrogenase.

This document also features a composition comprising 4-hydroxybutyrate, bio 1,4 butanediol, and a carboxylate reductase and an alcohol dehydrogenase. The composition can be cellular or acellular.

This document also features a composition comprising 4-hydroxybutyrate, bio putrescine, and a carboxylate reductase, an alcohol dehydrogenase, and at least one ω-transaminase. The composition can be cellular or acellular.

In another aspect, this document features a bio 1,4 butanediol produced by the method of enzymatically converting 4-hydroxybutyrate to 1,4 butanediol using a carboxylate reductase and an alcohol dehydrogenase.

This document also features a bio putrescine produced by the method of enzymatically converting 4-hydroxybutyrate to putrescine using a carboxylate reductase, an alcohol dehydrogenase, and at least one ω-transaminase.

The reactions of the pathways described herein can be performed in one or more cell (e.g., host cell) strains (a) naturally expressing one or more relevant enzymes, (b) genetically engineered to express one or more relevant enzymes, or (c) naturally expressing one or more relevant enzymes and genetically engineered to express one or more relevant enzymes. Alternatively, relevant enzymes can be extracted from any of the above types of host cells and used in a purified or semi-purified form. Extracted enzymes optionally can be immobilized to the floors and/or walls of appropriate reaction vessels. Moreover, such extracts include lysates (e.g. cell lysates), and partially purified lysates, that can be used as sources of relevant enzymes. In the methods provided by the document, all the steps can be performed in cells (e.g., host cells), all the steps can be performed using extracted enzymes, or some of the steps can be performed in cells and others can be performed using extracted enzymes. In any of the methods, the reaction may be a single step conversion in which one compound is directly converted to a different compound of interest (e.g., L-glutamate to 4-aminobutyrate), or the conversion may include two or more steps to convert one compound to a different compound.

Many of the enzymes described herein catalyze reversible reactions, and the reaction of interest may be the reverse of the described reaction. The schematic pathways shown in FIGS. 1 to 4 illustrate the reaction of interest for each of the intermediates.

In one aspect, this document features a method for producing a bioderived four carbon compound. The method for producing a bioderived four carbon compound can include culturing or growing a recombinant host as described herein under conditions and for a sufficient period of time to produce the bioderived four carbon compound, wherein, optionally, the bioderived four carbon compound is selected from the group consisting of 4-hydroxybutyrate, 4-aminobutyrate, putrescine, and 1,4-butanediol, and combinations thereof.

In one aspect, this document features composition comprising a bioderived four carbon compound as described herein and a compound other than the bioderived four carbon compound, wherein the bioderived four carbon compound is selected from the group consisting of 4-hydroxybutyrate, 4-aminobutyrate, putrescine, and 1,4-butanediol, and combinations thereof. For example, the bioderived four carbon compound is a cellular portion of a host cell or an organism.

This document also features a biobased polymer comprising the b4-hydroxybutyrate, 4-aminobutyrate, putrescine, and 1,4-butanediol, and combinations thereof.

This document also features a biobased resin comprising the 4-hydroxybutyrate, 4-aminobutyrate, putrescine, and 1,4-butanediol, and combinations thereof, as well as a molded product obtained by molding a biobased resin.

In another aspect, this document features a process for producing a biobased polymer that includes chemically reacting the bioderived 4-hydroxybutyrate, 4-aminobutyrate, putrescine, and 1,4-butanediol, with itself or another compound in a polymer producing reaction.

In another aspect, this document features a process for producing a biobased resin that includes chemically reacting the bioderived 4-hydroxybutyrate, 4-aminobutyrate, putrescine, and 1,4-butanediol, with itself or another compound in a resin producing reaction.

One of skill in the art understands that compounds containing carboxylic acid groups (including, but not limited to, organic monoacids, hydroxyacids, aminoacids, and dicarboxylic acids) are formed or converted to their ionic salt form when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base.

Acceptable organic bases include, but are not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. A salt of the present invention is isolated as a salt or converted to the free acid by reducing the pH to below the pKa, through addition of acid or treatment with an acidic ion exchange resin.

In a another aspect, the disclosure provides a non-naturally occurring organism comprising at least one exogenous nucleic acid encoding at least one polypeptide having the activity of at least one enzyme depicted in any one of FIGS. 1 to 5.

In a another aspect, the disclosure provides a nucleic acid construct or expression vector comprising (a) a polynucleotide encoding a polypeptide having the activity of a glutamate decarboxylase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a glutamate decarboxylase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 14 or SEQ ID NO: 19; or (b) a polynucleotide encoding a polypeptide having the activity of a carboxylate reductase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a carboxylate reductase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO: 18; or (c) a polynucleotide encoding a polypeptide having the activity of ω-transaminase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of ω-transaminase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11; or (d) a polynucleotide encoding a polypeptide having the activity of a phosphopantetheinyl transferase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having phosphopantetheinyl transferase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 12 or 13; or (e) a polynucleotide encoding a polypeptide having the activity of a decarboxylase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a decarboxylase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 15, 16 or 17. The disclosure further provides a composition comprising the nucleic acid construct or expression vector as recited above.

One of skill in the art understands that compounds containing amine groups (including, but not limited to, organic amines, aminoacids, and diamines) are formed or converted to their ionic salt form, for example, by addition of an acidic proton to the amine to form the ammonium salt, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids including, but not limited to, acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like. Acceptable inorganic bases include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. A salt of the present invention is isolated as a salt or converted to the free amine by raising the pH to above the pKb through addition of base or treatment with a basic ion exchange resin.

One of skill in the art understands that compounds containing both amine groups and carboxylic acid groups (including, but not limited to, aminoacids) are formed or converted to their ionic salt form by either 1) acid addition salts, formed with inorganic acids including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids including, but not limited to, acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like. Acceptable inorganic bases include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like, or 2) when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases include, but are not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. A salt can of the present invention is isolated as a salt or converted to the free acid by reducing the pH to below the pKa through addition of acid or treatment with an acidic ion exchange resin.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims. The word “comprising” in the claims may be replaced by “consisting essentially of” or with “consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of exemplary biochemical pathways leading to 4-hydroxybutyrate using 2-oxoglutarate acid as a central metabolite.

FIG. 2 is a schematic of an exemplary biochemical pathway leading to 4-aminobutyrate using 2-oxoglutarate acid as a central precursor.

FIG. 3 is a schematic of exemplary biochemical pathways leading to putrescine using 4-aminobutyrate, 4-hydroxybutyrate, succinate semialdehyde, or 1,4-butanediol as a central precursor.

FIG. 4 is a schematic of an exemplary biochemical pathway leading to 1,4 butanediol using 4-hydroxybutyrate as a central precursor.

FIG. 5 contains the amino acid sequences of a Mycobacterium marinum carboxylate reductase (see Genbank Accession No. ACC40567.1, SEQ ID NO: 1), a Mycobacterium smegmatis carboxylate reductase (see Genbank Accession No. ABK71854.1, SEQ ID NO: 2), a Segniliparus rugosus carboxylate reductase (see Genbank Accession No. EFV11917.1, SEQ ID NO: 3), a Mycobacterium massiliense carboxylate reductase (see Genbank Accession No. EIV11143.1, SEQ ID NO: 4), a Segniliparus rotundus carboxylate reductase (see Genbank Accession No. ADG98140.1, SEQ ID NO: 5), a Chromobacterium violaceum ω-transaminase (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 6), a Pseudomonas aeruginosa ω-transaminase (see Genbank Accession No. AAG08191.1, SEQ ID NO: 7), a Pseudomonas syringae ω-transaminase (see Genbank Accession No. AAY39893.1, SEQ ID NO: 8), a Rhodobacter sphaeroides ω-transaminase (see Genbank Accession No. ABA81135.1, SEQ ID NO: 9), an Escherichia coli ω-transaminase (see Genbank Accession No. AAA57874.1, SEQ ID NO: 10), a Vibrio fluvialis ω-transaminase (See Genbank Accession No. AEA39183.1, SEQ ID NO: 11), a Bacillus subtilis phosphopantetheinyl transferase (see Genbank Accession No. CAA44858.1, SEQ ID NO: 12), a Nocardia sp. NRRL 5646 phosphopantetheinyl transferase (see Genbank Accession No. AB183656.1, SEQ ID NO: 13), an Escherichia coli L-glulamate decarboxylase (see Genbank Accession No. AAA23833.1 (SEQ ID NO: 14) & AAA23834.1 (SEQ ID NO: 19)), a Lactococcus lactis α-ketoisovalerate decarboxylase (see Genbank Accession No. ADA65057.1, SEQ ID NO: 15), a Mycobacterium smegmalis 2-oxoglutarale decarboxylase (see Genbank Accession No. ABK74238.1, SEQ ID NO:16), a Salmonella typhimurium indolepyruvate decarboxylase (see Genbank Accession No. AHX78209.1, SEQ ID NO: 17) or a Mycobacterium smegmatis carboxylate reductase (see Genbank Accession No. ABK75684.1, SEQ ID NO:18).

FIG. 6 is a bar graph summarizing the change in absorbance at 340 nm after 1 hour, which is a measure of the consumption of NADPH and activity of three carboxylate reductase preparations in enzyme only controls (no substrate).

FIG. 7 is a bar graph of the change in absorbance at 340 nm after 1 hour, which is a measure of the consumption of NADPH and the activity of three carboxylate reductase preparations for converting 4-hydroxybutyrate to 4-hydroxybutanal relative to the empty vector control.

DETAILED DESCRIPTION

This document provides enzymes, non-natural pathways, cultivation strategies, feedstocks, host microorganisms and attenuations to the host's biochemical network, which generates a four carbon chain backbone via decarboxylation of central metabolites such as 2-oxoglutarate or L-glutamate and in which one or two terminal functional groups may be formed leading to the synthesis of one or more of 4-hydroxybutyrate, 4-aminobutyrate, putrescine (also known as tetramethylenediamine), and 1,4-butanediol. Succinate semialdehyde can be produced as an intermediate to other products. As used herein, the term “central precursor” is used to denote any metabolite in any metabolic pathway shown herein leading to the synthesis of a C4 building block. The term “central metabolite” is used herein to denote a metabolite that is produced in all microorganisms to support growth.

Host microorganisms described herein can include endogenous pathways that can be manipulated such that one or more C4 building blocks can be produced. In an endogenous pathway, the host microorganism naturally expresses all of the enzymes catalyzing the reactions within the pathway. A host microorganism containing an engineered pathway does not naturally express all of the enzymes catalyzing the reactions within the pathway but has been engineered such that all of the enzymes within the pathway are expressed in the host.

The term “exogenous” as used herein with reference to a nucleic acid (or a protein) and a host refers to a nucleic acid that does not occur in (and cannot be obtained from) a cell of that particular type as it is found in nature or a protein encoded by such a nucleic acid. Thus, a non-naturally-occurring nucleic acid is considered to be exogenous to a host once in the host. It is important to note that non-naturally-occurring nucleic acids can contain nucleic acid subsequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a host cell once introduced into the host, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acid since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally-occurring nucleic acid. A nucleic acid that is naturally-occurring can be exogenous to a particular host microorganism. For example, an entire chromosome isolated from a cell of yeast x is an exogenous nucleic acid with respect to a cell of yeast y once that chromosome is introduced into a cell of yeast y.

In contrast, the term “endogenous” as used herein with reference to a nucleic acid (e.g., a gene) (or a protein) and a host refers to a nucleic acid (or protein) that does occur in (and can be obtained from) that particular host as it is found in nature. Moreover, a cell “endogenously expressing” a nucleic acid (or protein) expresses that nucleic acid (or protein) as does a host of the same particular type as it is found in nature. Moreover, a host “endogenously producing” or that “endogenously produces” a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as does a host of the same particular type as it is found in nature.

For example, depending on the host and the compounds produced by the host, one or more of the following enzymes may be expressed in the host including a glutamate synthase, a glutamate decarboxylase, a 2-oxoghlarate decarboxylase, a 5-hydroxyvalerate dehydrogenase, an alcohol dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a ω-transaminase, or a carboxylate reductase. In recombinant hosts expressing a carboxylate reductase, aphosphopantetheinyl transferase also can be expressed as it enhances activity of the carboxylate reductase.

In some embodiments, a recombinant host includes an exogenous glutamate synthase and produces L-glutamate.

In some embodiments, a recombinant host includes an exogenous glutamate synthase and an exogenous glutamate decarboxylase and produces 4-aminobutyrate.

In some embodiments, a recombinant host includes an exogenous glutamate decarboxylase and produces 4-aminobutyrate.

In some embodiments, a recombinant host producing 4-aminobutyrate includes at least one exogenous nucleic acid encoding a ω-transaminase and further produces succinate semialdehyde. For example, a recombinant host can include an exogenous glutamate decarboxylase, an exogenous ω-transaminase, and an optional an exogenous glutamate synthase, and further produce succinate semialdehyde.

In some embodiments, a recombinant host producing 4-aminobutyrate can include at least one exogenous nucleic acid encoding a ω-transaminase and a dehydrogenase such as a 4-hydroxybutyrate dehydrogenase or a 5-hydroxyvalerate dehydrogenase, and further produce 4-hydroxybuytrate. For example, a recombinant host can include an exogenous glutamate decarboxylase, an exogenous ω-transaminase, an exogenous dehydrogenase, and an optional exogenous glutamate synthase, and further produce 4-hydroxybutyrate.

In some embodiments, a recombinant host can include an exogenous 2-oxoglutarate decarboxylase and an exogenous dehydrogenase such as a 4-hydroxybutyrate dehydrogenase or a 5-hydroxyvalerate dehydrogenase, and produce 4-hydroxybutyrate. Such a host further can include an exogenous carboxylate reductase and an exogenous alcohol dehydrogenase, and further produce 1,4-butanediol.

A recombinant host producing 4-aminobutyrate, 4-hydroxybutyrate, or succinate semialdehyde can include one or more of an exogenous carboxylate reductase, an exogenous co transaminase, or an exogenous alcohol dehydrogenase, and one or more (e.g., one, two, or three) optional exogenous enzymes such as a decarboxylase, dehydrogenase and/or a synthase, and produce putrescine.

In some embodiments, a recombinant host can include each of an exogenous carboxylate reductase and an exogenous ω-transaminase and produce putrescine. In some embodiments, a recombinant host can include each of an exogenous carboxylate reductase, an exogenous ω-transaminase, and an exogenous glutamate decarboxylase and produce putrescine.

In some embodiments, a recombinant host can include each of an exogenous carboxylate reductase, an exogenous ω-transaminase, an exogenous glutamate synthase, and an exogenous glutamate decarboxylase and produce putrescine.

In some embodiments, a recombinant host can include each of an exogenous carboxylate reductase, at least one exogenous ω-transaminase (e.g., two different exogenous ω-transaminases), an exogenous alcohol dehydrogenase, and produce putrescine.

In some embodiments, a recombinant host can include each of an exogenous carboxylate reductase, at least one exogenous ω-transaminase (e.g., two or three different exogenous ω-transaminases), an exogenous alcohol dehydrogenase, an exogenous 4-hydroxybutyrate dehydrogenase or an exogenous 5-hydroxyvalerate dehydrogenase, and an exogenous 2-oxoglutarate decarboxylase and produce putrescine.

In some embodiments, a recombinant host can include each of an exogenous carboxylate reductase, at least one exogenous ω-transaminase (e.g., two or three different exogenous ω-transaminases), an exogenous alcohol dehydrogenase, an exogenous 4-hydroxybutyrate dehydrogenase or an exogenous 5-hydroxyvalerate dehydrogenase, an exogenous glutamate decarboxylase, and an optional glutamate synthase, and produce putrescine.

A recombinant host producing 4-hydroxybutyrate can include one or more of a carboxylate reductase and an alcohol dehydrogenase, and produce 1,4-butanediol. A recombinant host producing 1,4-butanediol can include at least one exogenous ω-transaminase (e.g., one exogenous ω-transaminase or two different exogenous ω-transaminases) and optional second and/or third exogenous alcohol dehydrogenases and produce putrescine.

Within an engineered pathway, the enzymes can be from a single source, i.e., from one species or genus, or can be from multiple sources, i.e., different species or genera. Nucleic acids encoding the enzymes described herein have been identified from various organisms and are readily available in publicly available databases such as GenBank or EMBL.

As used herein, references to a particular enzyme (e.g. ω-transaminase) means a polypeptide having the activity of the particular enzyme (e.g. a polypeptide ω-transaminase activity).

Any of the enzymes described herein that can be used for production of one or more C4 building blocks can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of the corresponding wild-type enzyme. It will be appreciated that the sequence identity can be determined on the basis of the mature enzyme (e.g., with any signal sequence removed) or on the basis of the immature enzyme (e.g., with any signal sequence included). It also will be appreciated that the initial methionine residue may or may not be present on any of the enzyme sequences described herein.

For example, a carboxylate reductase described herein can have at least 70%/sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Mycobacterium marinum (see Genbank Accession No. ACC40567.1, SEQ ID NO: 1), a Mycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQ ID NO: 2), a Segniliparus rugosus (see Genbank Accession No. EFV11917.1, SEQ ID NO: 3), a Mycobacterium massiliense (see Genbank Accession No. EIV11143.1, SEQ ID NO: 4), a Segniliparus rotundus (see Genbank Accession No. ADG98140.1, SEQ ID NO: 5), or a Mycobacterium smegmalis (see Genbank Accession No. ABK75684.1, SEQ ID NO: 18) carboxylate reductase. See, FIG. 5.

For example, a ω-transaminase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Chromobacterium violaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 6), a Pseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO: 7), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ ID NO: 8), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1, SEQ ID NO: 9), an Escherichia coli (see Genbank Accession No. AAA57874.1, SEQ ID NO: 10), or a Vibrio fluvialis (see Genbank Accession No. AEA39183.1, SEQ ID NO: 11) ω-transaminase. Some of these ω-transaminases are diamine ω-transaminases. See, FIG. 5.

For example, a phosphopantetheinyl transferase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Bacillus subtilis phosphopantetheinyl transferase (see Genbank Accession No. CAA44858.1, SEQ ID NO: 12) or a Nocardia sp. NRRL 5646 phosphopanietheinyl transferase (see Genbank Accession No. ABI83656.1, SEQ ID NO:13). See FIG. 5.

For example, a decarboxylase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Escherichia coli L-glutamate decarboxylase (see Genbank Accession No. AAA23833.1 (SEQ ID NO: 14) & AAA23834.1 (SEQ ID NO: 19)), a Lactococcus lactis α-ketoisovalerate decarboxylase (see Genbank Accession No. ADA65057.1, SEQ ID NO: 15), a Mycobacterium smegmatis 2-oxoglutarate decarboxylase (see Genbank Accession No. ABK74238.1, SEQ ID NO:16), or a Salmonella typhimurium indolepyruvate decarboxylase (see Genbank Accession No. AHX78209.1, SEQ ID NO: 17). See FIG. 5.

The percent identity (homology) between two amino acid sequences can be determined as follows. First, the amino acid sequences are aligned using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site (e.g., worldwide web at .fr.com/blast/) or the U.S. government's National Center for Biotechnology Information web site (worldwide web at ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. Bl2seq performs a comparison between two amino acid sequences using the BLASTP algorithm. To compare two amino acid sequences, the options of Bl2seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share homology (identity), then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology (identity), then the designated output file will not present aligned sequences. Similar procedures can be following for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity (homology) is determined by dividing the number of matches by the length of the full-length polypeptide amino acid sequence followed by multiplying the resulting value by 100. It is noted that the percent identity (homology) value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It also is noted that the length value will always be an integer.

It will be appreciated that a number of nucleic acids can encode a polypeptide having a particular amino acid sequence. The degeneracy of the genetic code is well known to the art; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. For example, codons in the coding sequence for a given enzyme can be modified such that optimal expression in a particular species (e.g., bacteria or fungus) is obtained, using appropriate codon bias tables for that species.

Functional fragments of any of the enzymes described herein can also be used in the methods of the document. The term “functional fragment” as used herein refers to a peptide fragment of a protein that has at least 25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%; 98%; 99%; 100%; or even greater than 100%) of the activity of the corresponding mature, full-length, wild-type protein. The functional fragment can generally, but not always, be comprised of a continuous region of the protein, wherein the region has functional activity.

This document also provides (i) functional variants of the enzymes used in the methods of the document and (ii) functional variants of the functional fragments described above. Functional variants of the enzymes and functional fragments can contain additions, deletions, or substitutions relative to the corresponding wild-type sequences. Enzymes with substitutions will generally have not more than 50 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservative substitutions). This applies to any of the enzymes described herein and functional fragments. A conservative substitution is a substitution of one amino acid for another with similar characteristics. Conservative substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a nonconservative substitution is a substitution of one amino acid for another with dissimilar characteristics.

Deletion variants can lack one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid segments (of two or more amino acids) or non-contiguous single amino acids. Additions (addition variants) include fusion proteins containing: (a) any of the enzymes described herein or a fragment thereof; and (b) internal or terminal (C or N) irrelevant or heterologous amino acid sequences. In the context of such fusion proteins, the term “heterologous amino acid sequences” refers to an amino acid sequence other than (a). A heterologous sequence can be, for example a sequence used for purification of the recombinant protein (e.g., FLAG, polyhistidine (e.g., hexahistidine), hemagglutinin (HA), glutathione-S-transferase (GST), or maltosebinding protein (MBP)). Heterologous sequences also can be proteins useful as detectable markers, for example, luciferase, green fluorescent protein (GFP), or chloramphenicol acetyl transferase (CAT). In some embodiments, the fusion protein contains a signal sequence from another protein. In certain host cells (e.g., yeast host cells), expression and/or secretion of the target protein can be increased through use of a heterologous signal sequence. In some embodiments, the fusion protein can contain a carrier (e.g., KLH) useful, e.g., in eliciting an immune response for antibody generation) or ER or Golgi apparatus retention signals. Heterologous sequences can be of varying length and in some cases can be a longer sequences than the full-length target proteins to which the heterologous sequences are attached.

Engineered hosts can naturally express none or some (e.g., one or more, two or more, three or more, four or more, five or more, or six or more) of the enzymes of the pathways described herein in FIG. 1, 2, 3, or 4. Thus, a pathway within an engineered host can include all exogenous enzymes, or can include both endogenous and exogenous enzymes. Endogenous genes of the engineered hosts also can be disrupted to prevent the formation of undesirable metabolites or prevent the loss of intermediates in the pathway through other enzymes acting on such intermediates. Engineered hosts can be referred to as recombinant hosts or recombinant host cells. As described herein recombinant hosts can include nucleic acids encoding one or more of a reductase, decarboxylase, synthase, dehydrogenase, or ω-transaminase as described herein.

In addition, the production of one or more C4 building blocks can be performed in vitro using the isolated enzymes described herein, using a lysate (e.g., a cell lysate) from a host microorganism as a source of the enzymes, or using a plurality of lysates from different host microorganisms as the source of the enzymes.

Enzymes Generating the Terminal Hydroxyl Group in the Biosynthesis of a C4 Building Block

As depicted in FIGS. 1 and 4, a terminal hydroxyl group can be enzymatically formed using a dehydrogenase such as an alcohol dehydrogenase, a 5-hydroxyvalerate dehydrogenase, or a 4-hydroxybutyrate dehydrogenase.

For example, a terminal hydroxyl group leading to the synthesis of 4-hydroxybutyrate can be enzymatically formed by a dehydrogenase classified, for example, under EC 1.1.1.—such as a 5-hydroxyvalerate dehydrogenase (also known as 5-hydroxypentanoate dehydrogenase), for example, the gene product of CpnD (see, for example, Iwaki et al., 2002, Appl. Environ. Microbiol., 68(11):5671-5684), a 5-hydroxyvalerate dehydrogenase from Clostridium viride, or a 4-hydroxybutyrate dehydrogenase classified under EC 1.1.1.61 such as gbd (see, for example, Lütke-Eversloh & Steinbüchel, 1999, FEMS Microbiology Letters, 181(1):63-71). See, FIG. 1.

A terminal hydroxyl group leading to the synthesis of 1,4 butanediol can be enzymatically formed by an alcohol dehydrogenase classified under EC 1.1.1.—(e.g., EC 1.1.1.1, 1.1.1.2, 1.1.1.21, or 1.1.1.184). See, FIG. 4.

Enzymes Generating the Terminal Amine Groups in the Biosynthesis of a C4 Building Block

As depicted in FIGS. 1-3, terminal amine groups can be enzymatically formed using a ω-transaminase or a glutamate decarboxylase.

In some embodiments, one terminal amine group is enzymatically formed by a glutamate decarboxylase classified, for example, under EC 4.1.1.15, producing 4-aminobutyrate. See, FIGS. 1 and 2.

In some embodiments, one terminal amine group leading to the synthesis of putrescine can be enzymatically formed by a ω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as that obtained from Chromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO: 6), Pseudomonas aeruginosa (Genbank Accession No. AAG08191.1, SEQ ID NO: 7), Pseudomonas syringae (Genbank Accession No. AAY39893.1, SEQ ID NO: 8), Rhodobacter sphaeroides (Genbank Accession No. ABA81135.1, SEQ ID NO: 9), Vibrio fluvialis (Genbank Accession No. AEA39183.1, SEQ ID NO: 11), Streptomyces griseus, or Clostridium viride. An additional ω-transaminase that can be used in the methods and hosts described herein is from Escherichia coli (Genbank Accession No. AAA57874.1, SEQ ID NO: 10). Some of the ω-transaminases classified, for example, under EC 2.6.1.29 or EC 2.6.1.82 are diamine ω-transaminases (e.g., SEQ ID NO: 8). See, FIG. 3.

The reversible ω-transaminase from Chromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO: 7) has demonstrated activity accepting 4-aminobutyric acid as amino donor, thus forming the first terminal amine group in succinate semialdehyde (Kaulmann et al., Enzyme and Microbial Technology, 2007, 41, 628-637).

The reversible 4-aminobutyrate:2-oxoglutarate transaminase from Streptomyces griseus has been characterized (Yonaha et al., Eur. J. Biochem., 1985, 146, 101-106).

In some embodiments, the second terminal amine group leading to the synthesis of putrescine is enzymatically formed by a diamine transaminase. For example, the second terminal amino group can be enzymatically formed by a diamine transaminase classified, for example, under EC 2.6.1.29 or EC 2.6.1.76, or classified, for example, under EC 2.6.1.82, such as the gene product of YgjG from E. coli (Genbank Accession No. AAA57874.1, SEQ ID NO: 10).

The gene product of ygjG accepts a broad range of diamine carbon chain length substrates, including putrescine (Samsonova et al., BMC Microbiology, 2003, 3:2).

The diamine transaminase from E. coli strain B has demonstrated activity for 1,4 diaminobutane (Kim, The Journal of Chemistry, 1964, 239(3), 783-786).

Biochemical Pathways Pathway to 4-Hydroxybutyrate

As depicted in FIG. 1, 2-oxoglutarate can be converted to L-glutamate by a glutamate synthase classified, for example, under EC 1.4.1.13; followed by conversion of L-glutamate to 4-aminobutyrate by a glutamate decarboxylase classified, for example, under EC 4.1.1.15 (see Genbank Accession No. AAA23833.1 (SEQ ID NO: 14) & AAA23834.1 (SEQ ID NO: 19)); followed by conversion of 4-aminobutyrate to succinate semialdehyde by a ω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, or EC 2.6.1.96; followed by conversion of succinate semialdehyde to 4-hydroxybutyrate by a dehydrogenase classified, for example, under EC 1.1.1.—such as EC 1.1.1.61 (e.g., the gene product of gbd) or the gene product of cpnD.

As depicted in FIG. 1, 2-oxoglutarate can be converted to succinate semialdehyde using a 2-oxoglutarate decarboxylase classified, for example, under EC 4.1.1.43, EC 4.1.1.71, or EC 4.1.1.73, followed by conversion of succinate semialdehyde to 4-hydroxybutyrate by a dehydrogenase classified, for example, under EC 1.1.1.—such as EC 1.1.1.61 (e.g., the gene product of gbd) or the gene product of cpnD).

Pathway to 4-Aminobutyrate Using 2-Oxoglutarate as a Central Precursor

As depicted in FIG. 1, 2-oxoglutarate can be converted to succinate semialdehyde by a decarboxylase classified, for example, under EC 4.1.1.43, EC 4.1.1.71 or EC 4.1.1.74; followed by conversion of succinate semialdehyde to 4-aminobutyrate by a ω-transaminase classified, for example, under EC 2.6.1.—. The decarboxylase can be obtained, for example, from Lactococcus lactis α-ketoisovalerate decarboxylase (see Genbank Accession No. ADA65057.1, SEQ ID NO: 15), from Mycobacterium smegmatis 2-oxoglutarate decarboxylase (see Genbank Accession No. ABK74238.1, SEQ ID NO: 16) or from Salmonella typhimurium indolepyruvate decarboxylase (see Genbank Accession No. AHX78209.1, SEQ ID NO: 17).

As depicted in FIGS. 1 and 2, 2-oxoglutarate can be converted to L-glutamate by a glutamate synthase classified, for example, under EC 1.4.1.13; followed by conversion of L-glutamate to 4-aminobutyrate by a glutamate decarboxylase classified, for example, under EC 4.1.1.15.

Pathway Using 4-Aminobutyrate, 4-Hydroxybutyrate, Succinate Semialdehyde or 1,4-Butanediol as Central Precursor to Putrescine

In some embodiments, putrescine is synthesized from the central precursor 4-aminobutyrate (which can be produced, for example, as described in FIG. 2) by conversion of 4-aminobutyrate to 4-aminobutanal by a carboxylate reductase classified, for example, under EC 1.2.99.6 such as the gene product of car in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia, SEQ ID NOs: 12 and 13, respectively) or the gene products of GriC and GriD from Streptomyces griseus (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion of 4-aminobutanal to putrescine by a ω-transaminase (e.g., EC 2.6.1.—such as one of SEQ ID NOs: 6, 7, 8, or 10). The carboxylate reductase can be obtained, for example, from Mycobacterium marinum (Genbank Accession No. ACC40567.1, SEQ ID NO: 1), Mycobacterium smegmatis (Genbank Accession No. ABK71854.1, SEQ ID NO: 2), Segniliparus rugosus (Genbank Accession No. EFV11917.1, SEQ ID NO: 3), Mycobacterium massiliense (Genbank Accession No. EIV11143.1, SEQ ID NO: 4), Segniliparus rotundus (Genbank Accession No. ADG98140.1, SEQ ID NO: 5), or Mycobacterium smegmatis (see Genbank Accession No. ABK75684.1, SEQ ID NO: 18). See FIG. 3.

The carboxylate reductase encoded by the gene product of car and enhancer npt or sfp has broad substrate specificity, including terminal difunctional C4 and C5 carboxylic acids (Venkitasubramanian et al., Enzyme and Microbial Technology, 2008, 42, 130-137).

In some embodiments, putrescine is synthesized from the central precursor 4-hydroxybutyrate (which can be produced, for example, as described in FIG. 1), by conversion of 4-hydroxybutyrate to 4-hydroxybutanal by a carboxylale reductase classified, for example, under EC 1.2.99.6 such as the gene product of car (see above, e.g., one of SEQ ID NOs. 1-5) in combination with a phosphopantetheine transferase enhancer (see above); followed by conversion of 4-hydroxybutanal to 4-aminobutanol by a ω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:6-11, see above; followed by conversion to 4-aminobutanal by an alcohol dehydrogenase classified, for example, under EC 1.1.1.—(e.g., EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the gene product of YMR318C or YqhD (Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1; followed by conversion to putrescine by a ω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as one of SEQ ID NOs: 6-8 and 10, see above. See FIG. 3.

In some embodiments, putrescine is synthesized from the central precursor succinate semialdehyde (also known as 4-oxobutanoate) by conversion of succinate semialdehyde to butanedial by a carboxylate reductase classified, for example, under EC 1.2.99.6 such as the gene product of car (see above, e.g., SEQ ID NO: 5) in combination with a phosphopantelheine transferase enhancer (see above); followed by conversion to 4-aminobutanal by a ω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48; followed by conversion of 4-aminobutanal to putrescine by a ω-transaminase classified, for example, under EC 2.6.1.—such as one of SEQ ID NOs: 6-8 and 10. See FIG. 3.

In some embodiments, putrescine is synthesized from the central precursor 1,4-butanediol (which can be produced, for example, as described in FIG. 4), by conversion of 1,4-butanediol to 4-hydroxybutanal by an alcohol dehydrogenase classified, for example, under EC 1.1.1.—such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the gene product of YIMR318C or YqhD (from E. coli, GenBank Accession No. AAA69178.1) (see, e.g., Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; or Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1 (from Geobacillus stearothermophilus); followed by conversion of 4-hydroxybutanal to 4-aminobutanol by a ω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:6-11, see above; followed by conversion to 4-aminobutanal by an alcohol dehydrogenase classified, for example, under EC 1.1.1.—(e.g., EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the gene product of YMR318C or YqhD (Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1; followed by conversion to putrescine by a ω-transaminase classified, for example, under EC 2.6.1.—such as one of SEQ ID NOs:6-8 or 10, see above. See FIG. 3.

Pathway to 1,4-Butanediol Using 4-Hydroxybutyrate as Central Precursor

As depicted in FIG. 4, 1,4 butanediol can be synthesized from the central precursor 4-hydroxybutyrate by conversion of 4-hydroxybutyrate to 4-hydroxybutanal by a carboxylate reductase classified, for example, under EC 1.2.99.6 such as the gene product of car (see above, e.g., one of SEQ ID NOs: 1-5) in combination with a phosphopantelheine transferase enhancer (see above); followed by conversion of 4-hydroxybutanal to 1,4 butanediol by an alcohol dehydrogenase classified, for example, under EC 1.1.1.—such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184 such as the gene product of YMR318C or YqhD (from E. coli, GenBank Accession No. AAA69178.1) (see, e.g., Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; or Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1 (from Geobacillus stearothermophilus). See, FIG. 5 for the amino acid sequences of the above proteins.

Cultivation Strategy

In some embodiments, a cultivation strategy entails either achieving an anaerobic, aerobic or micro-aerobic cultivation condition.

In some embodiments, the cultivation strategy entails nutrient limitation such as nitrogen, phosphate or oxygen limitation.

In some embodiments, a cell retention strategy using, for example, ceramic hollow fiber membranes can be employed to achieve and maintain a high cell density during either fed-batch or continuous fermentation.

In some embodiments, the principal carbon source fed to the fermentation in the synthesis of one or more C4 building blocks can derive from biological or non-biological feedstocks.

In some embodiments, the biological feedstock can be or can derive from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste.

The efficient catabolism of crude glycerol stemming from the production of biodiesel has been demonstrated in several microorganisms such as Escherichia coli, Cupriavidus necator, Pseudomonas oleavorans, Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem. Biotechnol., 2012, 166:1801-1813; Yang et al., Biotechnology for Biofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol., 2011, 90:885-893).

The efficient catabolism of lignocellulosic-derived levulinic acid has been demonstrated in several organisms such as Cupriavidus necator and Pseudomonas putida in the synthesis of 3-hydroxyvalerate via the precursor propanoyl-CoA (Jaremko and Yu, 2011, supra; Martin and Prather, J. Biotechnol., 2009, 139:61-67).

The efficient catabolism of lignin-derived aromatic compounds such as benzoate analogues has been demonstrated in several microorganisms such as Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinion in Biotechnology, 2011, 22, 394-400; Pérez-Pantoja et al., FEMS Microbiol. Rev., 2008, 32, 736-794).

The efficient utilization of agricultural waste, such as olive mill waste water has been demonstrated in several microorganisms, including Yarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008, 99(7):2419-2428).

The efficient utilization of fermentable sugars such as monosaccharides and disaccharides derived from cellulosic, hemicellulosic, cane and beet molasses, cassava, corn and other agricultural sources has been demonstrated for several microorganism such as Escherichia coli, Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcus lactis (see, e.g., Hermann et al, J. Biotechnol., 2003, 104:155-172; Wee et al., Food Technol. Biotechnol., 2006, 44(2): 163-172; Ohashi et al., J. Bioscience and Bioengineering, 1999, 87(5):647-654).

The efficient utilization of furfural, derived from a variety of agricultural lignocellulosic sources, has been demonstrated for Cupriavidus necator (Li et al., Biodegradation, 2011, 22:1215-1225).

In some embodiments, the non-biological feedstock can be or can derive from natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.

The efficient catabolism of methanol has been demonstrated for the methylotrophic yeast Pichia pastoris.

The efficient catabolism of ethanol has been demonstrated for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008, 105(6) 2128-2133).

The efficient catabolism of CO₂ and H₂, which may be derived from natural gas and other chemical and petrochemical sources, has been demonstrated for Cupriavidus necator (Prybylski et al., Energy, Sustainability and Society, 2012, 2:11).

The efficient catabolism of syngas has been demonstrated for numerous microorganisms, such as Clostridium ljungdahlii and Clostridium autoethanogenum (Kopke et al., Applied and Environmental Microbiology, 2011, 77(15):5467-5475).

The efficient catabolism of the non-volatile residue waste stream from cyclohexane processes has been demonstrated for numerous microorganisms, such as Delftia acidovorans and Cupriavidus necator (Ramsay et al., Applied and Environmental Microbiology, 1986, 52(1):152-156).

In some embodiments, the host microorganism is a prokaryote. For example, the prokaryote can be a bacterium from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; from the genus Delftia such as Delftia acidovorans; from the genus Bacillus such as Bacillus subtillis; from the genus Lactobacillus such as Lactobacillus delbrueckii; or from the genus Lactococcus such as Lactococcus lactis. Such prokaryotes also can be a source of genes to construct recombinant host cells described herein that are capable of producing one or more C4 building blocks.

In some embodiments, the host microorganism is a eukaryote. For example, the eukaryote can be a filamentous fungus, e.g., one from the genus Aspergillus such as Aspergillus niger. Alternatively, the eukaryote can be a yeast, e.g., one from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; or from the genus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkia such as Issathenkia orientalis; from the genus Debaryomyces such as Debaryomyces hansenii; from the genus Arxula such as Arxula adenoinivorans; or from the genus Kluyveromyces such as Kluyveromyces lactis. Such eukaryotes also can be a source of genes to construct recombinant host cells described herein that are capable of producing one or more C4 building blocks.

Metabolic Engineering

The present document provides methods involving less than all the steps described for all the above pathways. Such methods can involve, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of such steps. Where less than all the steps are included in such a method, the first, and in some embodiments the only, step can be any one of the steps listed.

Furthermore, recombinant hosts described herein can include any combination of the above enzymes such that one or more of the steps, e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more of such steps, can be performed within a recombinant host. This document provides host cells of any of the genera and species listed and genetically engineered to express one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, 11, 12 or more) recombinant forms of any of the enzymes recited in the document. Thus, for example, the host cells can contain exogenous nucleic acids encoding enzymes catalyzing one or more of the steps of any of the pathways described herein.

In addition, this document recognizes that where enzymes have been described as accepting CoA-activated substrates, analogous enzyme activities associated with [acp]-bound substrates exist that are not necessarily in the same enzyme class.

Also, this document recognizes that where enzymes have been described accepting (R)-enantiomers of substrate, analogous enzyme activities associated with (S)-enantiomer substrates exist that are not necessarily in the same enzyme class.

This document also recognizes that where an enzyme is shown to accept a particular co-factor, such as NADPH, or co-substrate, such as acetyl-CoA, many enzymes are promiscuous in terms of accepting a number of different co-factors or co-substrates in catalyzing a particular enzyme activity. Also, this document recognizes that where enzymes have high specificity for e.g., a particular co-factor such as NADH, an enzyme with similar or identical activity that has high specificity for the co-factor NADPH may be in a different enzyme class.

In some embodiments, the enzymes in the pathways outlined herein are the result of enzyme engineering via non-direct or rational enzyme design approaches with aims of improving activity, improving specificity, reducing feedback inhibition, reducing repression, improving enzyme solubility, changing stereo-specificity, or changing co-factor specificity.

In some embodiments, the enzymes in the pathways outlined here can be gene dosed, i.e., overexpressed, into the resulting genetically modified organism via episomal or chromosomal integration approaches.

In some embodiments, genome-scale system biology techniques such as Flux Balance Analysis can be utilized to devise genome scale attenuation or knockout strategies for directing carbon flux to a C4 building block.

Attenuation strategies include, but are not limited to; the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors and RNAi interference.

In some embodiments, fluxomic, metabolomic and transcriptomal data can be utilized to inform or support genome-scale system biology techniques, thereby devising genome scale attenuation or knockout strategies in directing carbon flux to a C4 building block.

In some embodiments, the host microorganism's tolerance to high concentrations of a C4 building block can be improved through continuous cultivation in a selective environment.

In some embodiments, the host microorganism's endogenous biochemical network can be attenuated or augmented to (1) ensure the intracellular availability of 2-oxoglutarate or glutamic acid, (2) create a co-factor imbalance that may only be balanced via the formation of one or more C4 building blocks, (3) prevent degradation of central metabolites, central precursors leading to and including one or more C4 building blocks and/or (4) ensure efficient efflux from the cell.

In some embodiments requiring the intracellular availability of 2-oxogluratate or L-glutamate for C4 building block synthesis, the enzymes catalyzing anaplerotic reactions supplementing the citric acid cycle intermediates are amplified, such as a phosphoenolpyruvate carboxylase or a pyruvate carboxylase.

In some embodiments, where pathways require excess NADH co-factor for C4 building block synthesis, a recombinant formate dehydrogenase gene can be overexpressed in the host organism (Shen et al., 2011, supra).

In some embodiments, where pathways require excess NADH or NADPH co-factor for C4 building block synthesis, a transhydrogenase can be attenuated.

In some embodiments, carbon flux can be directed into the pentose phosphate cycle to increase the supply of NADPH by attenuating an endogenous glucose-6-phosphate isomerase (EC 5.3.1.9).

In some embodiments, carbon flux can be redirected into the pentose phosphate cycle to increase the supply of NADPH by overexpression a 6-phosphogluconate dehydrogenase and/or a transketolase (Lee et al., 2003, Biotechnology Progress, 19(5), 1444-1449).

In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C4 building block, a gene such as UdhA encoding apuridine nucleotide transhydrogenase can be overexpressed in the host organisms (Brigham et al., Advanced Biofuels and Bioproducts, 2012, Chapter 39, 1065-1090).

In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C4 Building Block, a recombinant glyceraldehyde-3-phosphate-dehydrogenase gene such as GapN can be overexpressed in the host organisms (Brigham et al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C4 building block, a recombinant malic enzyme gene such as maeA or maeB can be overexpressed in the host organisms (Brigham et al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C4 building block, a recombinant glucose-6-phosphate dehydrogenase gene such as zwf can be overexpressed in the host organisms (Lim et al., J. Bioscience and Bioengineering, 2002, 93(6), 543-549).

In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C4 building block, a recombinant fructose 1.6 diphosphatase gene such as fbp can be overexpressed in the host organisms (Becker et al., J. Biotechnol., 2007, 132:99-109).

In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C4 building block, endogenous triose phosphate isomerase (EC 5.3.1.1) can be attenuated.

In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C4 building block, a recombinant glucose dehydrogenase such as the gene product of gdh can be overexpressed in the host organism (Satoh et al., J. Bioscience and Bioengineering, 2003, 95(4):335-341).

In some embodiments, endogenous enzymes facilitating the conversion of NADPH to NADH can be attenuated, such as the NADH generation cycle that may be generated via inter-conversion of glutamate dehydrogenases classified under EC 1.4.1.2 (NADH-specific) and EC 1.4.1.4 (NADPH-specific).

In some embodiments, an endogenous glutamate dehydrogenase (EC 1.4.1.3) that utilizes both NADH and NADPH as co-factors can be attenuated.

In some embodiments using hosts that naturally accumulate polyhydroxyalkanoates, the endogenous polyhydroxyalkanoate synthase enzymes can be attenuated in the host strain.

In some embodiments, a L-alanine dehydrogenase can be overexpressed in the host to regenerate L-alanine from pyruvate as an amino donor for ω-transaminase reactions.

In some embodiments, a L-glutamate dehydrogenase, a L-glutamine synthetase, or a glutamate synthase can be overexpressed in the host to regenerate L-glutamate from 2-oxoglutarate as an amino donor for ω-transaminase reactions.

In some embodiments, the efflux of a C4 building block across the cell membrane to the extracellular media can be enhanced or amplified by genetically engineering structural modifications to the cell membrane or increasing any associated transporter activity for a C4 building block.

The efflux of putrescine can be enhanced or amplified by overexpressing broad substrate range multidrug transporters such as Bit from Bacillus subtilis (Woolridge et al., 1997, J. Biol. Chem., 272(14):8864-8866); AcrB and AcrD from Escherichia coli (Elkins & Nikaido, 2002, J. Bacteriol., 184(23), 6490-6499), NorA from Staphylococcus aereus (Ng et al., 1994, Antimicrob Agents Chemother, 38(6), 1345-1355), or Bmr from Bacillus subtilis (Neyfakh, 1992, Antimicrob Agents Chemother, 36(2), 484-485).

The efflux of 4-aminobutyrate and putrescine can be enhanced or amplified by overexpressing the solute transporters such as the lysE transporter from Corynebacterium glutamicum (Bellmann et al., 2001, Microbiology, 147, 1765-1774).

Producing C4 Building Blocks Using a Recombinant Host

Typically, one or more C4 building blocks can be produced by providing a host microorganism and culturing the provided microorganism with a culture medium containing a suitable carbon source as described above. In general, the culture media and/or culture conditions can be such that the microorganisms grow to an adequate density and produce a C4 building block efficiently. For large-scale production processes, any method can be used such as those described elsewhere (Manual of Industrial Microbiology and Biotechnology, 2^(nd) Edition, Editors: A. L. Demain and J. E. Davies, ASM Press; and Principles of Fermentation Technology, P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g., a 100 gallon, 200 gallon, 500 gallon, or more tank) containing an appropriate culture medium is inoculated with a particular microorganism. After inoculation, the microorganism is incubated to allow biomass to be produced. Once a desired biomass is reached, the broth containing the microorganisms can be transferred to a second tank. This second tank can be any size. For example, the second tank can be larger, smaller, or the same size as the first tank. Typically, the second tank is larger than the first such that additional culture medium can be added to the broth from the first tank. In addition, the culture medium within this second tank can be the same as, or different from, that used in the first tank.

Once transferred, the microorganisms can be incubated to allow for the production of a C4 building block. Once produced, any method can be used to isolate C4 building blocks. For example, C4 building blocks can be recovered selectively from the fermentation broth via adsorption processes. In the case of 4-aminobutyrate, the resulting eluate can be further concentrated via evaporation, crystallized via evaporative and/or cooling crystallization, and the crystals recovered via centrifugation. In the case of putrescine and 1,4-butanediol, distillation may be employed to achieve the desired product purity.

EXAMPLES Example 1 Enzyme Activity of Carboxylate Reductase Using 4-Hydroxybutyrate as Substrate and Forming 4-Hydroxybutanal

A nucleotide sequence encoding a HIS-tag was added to the nucleic acid sequences from Mycobacterium smegmatis, Segniliparus rugosus and Segniliparus rotundus that encode the carboxylate reductases of SEQ ID NO: 18 (ABK75684.1), SEQ ID NO: 3 (EFV11917.1) and SEQ ID NO: 5 (ADG98140.1), respectively (see FIG. 5), such that N-terminal HIS tagged carboxylate reductases could be produced. Each of the modified genes was cloned into a pET Duet expression vector along with a sfp gene encoding a HIS-tagged phosphopantetheine transferase from Bacillus subtilis, both under the T7 promoter. Each expression vector was transformed into a BL21 [DE3] E. coli host. The resulting recombinant E. coli strains were cultivated in pre-culture containing 20 mL LB media and antibiotic selection pressure at 37° C., thereafter inoculating a 1 L shake flask containing 350 mL LB media with antibiotic selection pressure at 37° C., shaking at 200 rpm. The cultures were induced using 1 mM IPTG and each culture was cultivated overnight at 25° C.

The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication, and the cell debris was separated from the supernatant via centrifugation at 4° C. The carboxylate reductases and phosphopantetheine transferases were purified from the supernatant using Ni-affinity chromatography and buffer exchanged and concentrated into 50 mM potassium phosphate buffer (pH=6.8), 50 mM NaCl and 5% glycerol via ultrafiltration.

Enzyme activity assays (i.e., from γ-butyrolactone via 4-hydroxybutyrate to 4-hydroxybutanal) were performed in duplicate in a buffer composed of a final concentration of 50 mM potassium phosphate buffer (pH=6.8), 75 μM ZnCl₂, 1.25 mg/mL Acinetobacter sp SE19 lactonase, 10 mM γ-butyrolactone, 10 mM MgCl₂, 1 mM ATP and 0.5 mM NADPH. Each enzyme activity assay reaction was initiated by adding purified carboxylate reductase and phosphopantetheine transferase gene products or the empty vector control to the assay buffer containing the 4-hydroxybutyrate formed from γ-butyrolactone and then incubated at room temperature for 1 hour. The consumption of NADPH was monitored by absorbance at 340 nm. Each enzyme only control without γ-butyrolactone demonstrated low base line consumption of NADPH. See bars for ABK75684.1, EFV11917.1 and ADG98140.1 in FIG. 6.

The gene products of SEQ ID NOs: 18 (ABK75684.1), SEQ ID NO: 3 (EFV11917.1) and SEQ ID NO: 5 (ADG98140.1), enhanced by the gene product of sfp, accepted 4-hydroxybutyrate as substrate, as confirmed against the empty vector control (see FIG. 7), and synthesized 4-hydroxybutanal.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

1. A non-naturally occurring method of producing 1,4-butanediol, comprising enzymatically converting 4-hydroxybutyrate to 1,4 butanediol using a carboxylate reductase and an alcohol dehydrogenase.
 2. The method of claim 1, wherein 4-hydroxybutyrate is enzymatically synthesized from L-glutamate or 2-oxoglutarate.
 3. (canceled)
 4. The method of claim 2, wherein: 4-hydroxybutyrate is enzymatically synthesized from L-glutamate; L-glutamate is enzymatically converted to 4-aminobutyrate; 4-aminobutyrate is enzymatically converted to succinate semialdehyde; and succinate semialdehyde is enzymatically converted to 4-hydroxybutyrate.
 5. The method of claim 4, wherein L-glutamate is enzymatically converted to 4-hydroxybutyrate using: (i) a glutamate decarboxylase; (ii) a ω-transaminase; and (iii) a dehydrogenase selected from a 4-hydroxybutyrate dehydrogenase and a 5-hydroxyvalerate dehydrogenase.
 6. (canceled)
 7. The method of claim 2, wherein: 4-hydroxybutyrate is enzymatically synthesized from 2-oxoglutarate; and 2-oxoglutarate is enzymatically converted to succinate semialdehyde; and succinate semialdehyde is enzymatically converted to 4-hydroxybutyrate, or 2-oxoglutarate is enzymatically converted to L-glutamate; L-glutamate is enzymatically converted to 4-aminobutyrate; 4-aminobutyrate is enzymatically converted to succinate semialdehyde and succinate semialdehyde is enzymatically converted to 4-hydroxybutyrate
 8. (canceled)
 9. The method of claim 7, wherein 2-oxoglutarate is enzymatically converted to succinate semialdehyde using a 2-oxoglutarate decarboxylase, and optionally wherein succinate semialdehyde is enzymatically converted to 4-hydroxybutyrate using a 4-hydroxybutyrate dehydrogenase or a 5-hydroxyvalerate dehydrogenase. 10-13. (canceled)
 14. A method of producing putrescine, comprising: enzymatically converting 4-hydroxybutyrate to putrescine using a carboxylate reductase, an alcohol dehydrogenase, and at least one ω-transaminase; enzymatically converting 4-aminobutyrate to putrescine using a carboxylate reductase and a ω-transaminase; or enzymatically converting 1.4 butanediol to putrescine using at least one alcohol dehydrogenase and at least one ω-transaminase. 15-19. (canceled)
 20. The method of claim 1, wherein the method, in all or in part, is performed in a recombinant host by fermentation.
 21. The method of claim 20, wherein the recombinant host is subjected to a cultivation strategy under aerobic, anaerobic, or micro-aerobic cultivation conditions.
 22. The method of claim 20, wherein: the recombinant host is cultured under conditions of nutrient limitation; or the recombinant host is retained using a ceramic membrane.
 23. (canceled)
 24. The method of claim 20, wherein the principal carbon source fed to the fermentation derives from a biological feedstock.
 25. The method of claim 20, wherein: the principal carbon source fed to the fermentation is a biological feedstock that is, or derives from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste; or the principal carbon source fed to the fermentation is a non-biological feedstock that is, or derives from, natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.
 26. The method of claim 20, wherein the principal carbon source fed to the fermentation derives from a non-biological feedstock.
 27. (canceled)
 28. The method of claim 20, wherein the recombinant host is a prokaryote.
 29. The method of claim 20, wherein: the recombinant host is a prokaryote from a genus selected from Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus, and Rhodococcus; or the recombinant host is a eukaryote from a genus selected from Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, and Kluyveromyces.
 30. The method of claim 29, wherein: the prokaryote is selected from Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtillis, Lactobacillus delbrueckii, Lactococcus lactis, and Rhodococcus equi; or the eukaryote is selected from Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans, and Kluyveromyces lactis.
 31. The method of claim 20, wherein the recombinant host is a eukaryote.
 32. (canceled)
 33. (canceled)
 34. The method of claim 20, wherein: the recombinant host comprises one or more of the following attenuated enzymes: a polyhydroxyalkanoate synthase; a triose phosphate isomerase; a glucose-6-phosphate isomerase; a transhydrogenase; an NADH-specific glutamate dehydrogenase; or a NADH/NADPH-utilizing glutamate dehydrogenase; or the recombinant host overexpresses one or more genes encoding: a phosphoenolpyruvate carboxylase; a pyruvate carboxylase; a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a formate dehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose dehydrogenase; a glucose-6-phosphate dehydrogenase; a fructose 1.6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase; a L-glutamine synthetase; a lysine transporter; a dicarboxylate transporter and/or a multidrug transporter.
 35. (canceled)
 36. A recombinant host comprising: at least one exogenous nucleic acid encoding (i) a glutamate decarboxylase, (ii) an alcohol dehydrogenase selected from a 4-hydroxybutyrate dehydrogenase and a 5-hydroxyvalerate dehydrogenase, and (iii) a first exogenous ω-transaminase; or at least one exogenous nucleic acid encoding (i) a 2-oxoglutarate decarboxylase and (ii) an alcohol dehydrogenase selected from a 4-hydroxybutyrate dehydrogenase and a 5-hydroxyvalerate dehydrogenase, wherein the recombinant host produces 4-hydroxybutyrate.
 37. (canceled)
 38. The recombinant host of claim 36, further comprising: an exogenous carboxylate reductase, a second and a third exogenous ω-transaminase, and an exogenous alcohol dehydrogenase, wherein the recombinant host further produces putrescine; or an exogenous carboxylate reductase and an exogenous alcohol dehydrogenase, wherein the recombinant host further produces 1,4-butanediol. 39-50. (canceled)
 51. A composition comprising: 4-hydroxybutyrate; a bio-derived 1,4 butanediol or a bio-derived putrescine; and a carboxylate reductase, an alcohol dehydrogenase, and optionally at least one ω-transaminase.
 52. (canceled)
 53. The composition of claim 51, wherein the composition is acellular.
 54. The composition of claim 51, wherein the composition is cellular. 55-58. (canceled)
 59. A nucleic acid construct or expression vector comprising: (a) a polynucleotide encoding a polypeptide having the activity of a glutamate decarboxylase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a glutamate decarboxylase is selected from polypeptides having at least 70% sequence identity to the polypeptide of SEQ ID NO: 14 or SEQ ID NO: 19; (b) a polynucleotide encoding a polypeptide having the activity of a carboxylate reductase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a carboxylate reductase is selected from polypeptides having at least 70% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 18; (c) a polynucleotide encoding a polypeptide having the activity of a ω-transaminase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a ω-transaminase is selected from polypeptides having at least 70% sequence identity to the polypeptide of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11; (d) a polynucleotide encoding a polypeptide having the activity of a phosphopantetheinyl transferase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a phosphopantetheinyl transferase is selected from polypeptides having at least 70% sequence identity to the polypeptide of SEQ ID NO: 12 or 13; or (e) a polynucleotide encoding a polypeptide having the activity of a decarboxylase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a decarboxylase is selected from polypeptides having at least 70% sequence identity to the polypeptide of SEQ ID NO: 15, 16, or
 17. 60. (canceled) 